AU2010307466B2

AU2010307466B2 - Method for preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester

Info

Publication number: AU2010307466B2
Application number: AU2010307466A
Authority: AU
Inventors: Hui Ho Kim; Dae Won Lee; Dong Ho Lee; Hyun Seok Lee; Sang Chul Lim; Moo Yong Uhm
Original assignee: SK Biopharmaceuticals Co Ltd
Current assignee: SK Biopharmaceuticals Co Ltd
Priority date: 2009-10-15
Filing date: 2010-10-15
Publication date: 2015-02-05
Anticipated expiration: 2030-10-15
Also published as: WO2011046380A3; EP2488505A4; IL218787A0; ES2536488T3; US20110111467A1; US9434970B2; HUE025816T2; PL2488505T3; MX2012004055A; CN102574821B; RU2012119801A; CN102574821A; RU2539983C2; JP2013507911A; CL2012000959A1; HK1172625A1; BR112012008858B1; US8404461B2; US9068207B2; CA2776743C

Abstract

Disclosed is a method for the preparation of carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester, comprising the enantioselective enzyme reduction of the corresponding arylketone and the carbamation of the resultant alcohol.

Description

WO 20111046380 PCT/KR2010/007069 Description Title of Invention: METHOD FOR PREPARATION OF CARBAMIC ACID (R)-1-ARYL-2-TETRAZOLYL-ETHYL ESTER Technical Field [1] The present invention relates to a method for the preparation of carbamic acid (R)- 1 -aryl-2-tetrazolyl-ethyl ester. More particularly, the present invention relates to a method for preparing carbamic acid (R)-1-aryl-2-tetrazolyl-ethyl ester, comprising the enantioselective enzyme reduction of an arylketone. Background Art [2] As disclosed in U. S. Patent Application Publication No. 2006/0258718 Al, carbamic acid (R)- 1 -aryl-2-tetrazolyl-ethyl esters (hereinafter referred to as "the carbamate compounds") with anticonvulsant activity are useful in the treatment of disorders of the central nervous system, especially including anxiety, depression, convulsion, epilepsy, migraines, bipolar disorder, drug abuse, smoking, ADHD, obesity, sleep disorders, neuropathic pain, strokes, cognitive impairment, neurode generation, strokes and muscle spasms. [3] Depending on the position of N in the tetrazole moiety thereof, the carbamate compounds are divided into two positional isomers: tetrazole- 1-yl (hereinafter referred to as "IN tetrazole") and treatzole-2-yl (hereinafter referred to as "2N tetrazole"). The introduction of tetrazole for the preparation of the carbamate compounds results in a 1:1 mixture of the two positional isomers which are required to be individually isolated for pharmaceutical use. [4] Having chirality, the carbamate compounds must be in high optical purity as well as chemical purity as they are used as medications. [5] In this regard, U. S. Patent Application Publication No. 2006/0258718 Al uses the pure enantiomer (R)-aryl-oxirane as a starting material which is converted into an alcohol intermediate through a ring-opening reaction by tetrazole in the presence of a suitable base in a solvent, followed by introducing a carbamoyl group into the alcohol intermediate. For isolation and purification of the 1N and 2N positional isomers thus produced, column chromatography is set after the formation of an alcohol intermediate or carbamate. [6] For use in the preparation, (R)-2-aryl-oxirane may be synthesized from an optically active material, such as substituted (R)-mandelic acid derivative, via various routes or obtained by asymmetric reduction-ring formation reaction of a-halo arylketone or by separation of racemic 2-aryl-oxirane mixture into its individual enantiomers. As such, 2 (R-2ary-oxirane is an expensive compound. [7] In addition, the ring-open ug teactin of (R)-2-ary boxrane with tetrazNle is performed at relatively high temperaturs because of the low nucleophilicity of the tetrazole. However, hin opening reaction includes highly likely risk of a runaway reaction because tetrazoles start t spoftaleously degrade at 110 - (. 5 8 In terms of a action of rac.ton, as t are two reaction sites in each (R) 2 -. Ioxirane and tetrazole, the rtng-opening reaction therebetween affords the substitution of IN- or 2N-tetrazcde at the benzyl or terninal position, resElting in a mixture of a total of 4 positional isomers, Theretdfm, individual positional isomers are low in production yield and difficult to isolate atid purify. Disclosure of Invention 10 Technical Problem [9] One aspect of the present inven ton provides a novel method for preparing (R)- I-aryl-2-tetrazolyvl-ethyl ester. Solution to Problem [10] Disclosed herein is a method for preparing carbamic acid (R)-1-aryl-2-tetrazolyl ethyl ester, represented by Chemical Formula 1, comprising: subjecting an 15 arylketone, represented by Chemical Formula 2, to enantio selective enzymatic reduction; and carbamating an alcohol compound of (R)-configuration, represented by Chemical Formula 3: [11] [Chemical Formula 1] [12] 0 20 A', N RI 2 R2 [13] [Chemical Formula 2] 25 [14] 3 0 N-A R1 N R2 [15] [Chemical Formula 3] [16] OH NsA, 2 N [1,7] wherein. [18] R and R, are independently selected from a gIoup consisting of hydrogen, halogen, perfluoroalkyL alkyI of 1 to 8 carbon awoms, tikoaLkoxy of 1 to 8 carbon atoms, and alkoxy of 1 to 8 carton atoms; and 15 [19] one of A, and A. is CH with the other being N. [19a] According to an aspect of the present invention there is provided a method for preparing carbamic acid aryl-2-tetrazolyl ethyl ester, represented by Chemical Formula 1, comprising: subjecting an arylketone, represented by Chemical Formula 2, to enantioselective enzymatic reduction to form an alcohol compound 20 of (R)-configuration, represented by Chemical Formula 3; and carbamating said alcohol [Chemical Formula 1] 0

H

2 N 0 N:A 1 N R1 2 R2 3a [Chemical Formula 2] 0 N:Aj N RI

A

2 / R2 [Chemical Formula 3] OH N::Aj N N A RI A 5 wherein

R

1 and R 2 are independently selected from a group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; one of A 1 and A 2 is CH with the other being N; and 10 said enantioselective enzymatic reduction is carried out in a reaction mixture comprising said compound of Chemical Formula 2, an oxidoreductase having at least 60% homology with amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4, NADH or NADPH as a cofactor that is oxidized during the reduction process and continuously regenerated, a 15 cosubstrate comprising a secondary alcohol represented by the Formula RxRYCHOH, wherein Rx represents carbon with x being an integer from 1-10 and Ry represents hydrogen with y being an integer equal to two times the value of x plus two, and a suitable buffer. [19b] According to another aspect of the present invention there is provided a method 20 for preparing an alcohol compound, represented by the following Chemical Formula 3, through the enantioselective enzymatic reduction of an arylketone, represented by the following Chemical Formula 2: 3b [Chemical Formula 2] Ii N R24 [Chemical Formula 3] OH N~A A"N 5 wherein,

R

1 and R 2 are independently selected from a group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; one of Ai and A 2 is CH with the other being N; and 10 said reduction is carried out in a reaction mixture comprising said compound of Chemical Formula 2, an oxidoreductase having at least 60% homology with amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4, NADH or NADPH as a cofactor that is oxidized during the reduction process and continuously regenerated, a cosubstrate comprising a secondary 15 alcohol represented by the Formula RxRYCHOH, wherein Rx represents carbon with x being an integer from 1-10 and Ry represents hydrogen with y being an integer equal to two times the value of x plus two, and a suitable buffer. Advantageous Effects of Invention [20] According to one aspect of the present invention, carband compund of high opical purity and high chemical puri) Cal bn b nufa e comically. 20 Best Mode for' Catrrying out the Invention [211 Other aspect of the present invenion povidei a novel method for preparing an alcohol compyund. pasene by the following Chernical Fonrmla 3, thrmgh the enantioselective enzymatic reduction ot an aryketone, represented by the following Chemical Formula 2: [221 [Chemical FoRnula 2] 25 [231 WO 2011/046380 PCT/KR2010/007069 N R1 N 2 R2 [24] [Chemical Formula 3] [25] OH N--A \N NA //

A

2 R1 2 R2 [26] wherein, [27] R 1 and R 2 are independently selected from a group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; [28] one of A 1 and A2 is CH with the other being N; and [29] said reduction is carried out in a reaction mixture comprising said compound of Chemical Formula 2, an oxidoreductase having at least 60% homology with amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4, NADH or NADPH as a cofactor that is oxidized during the reduction process and con tinuously regenerated, a cosubstrate comprising a secondary alcohol represented by the Formula RxRyCHOH, wherein Rx represents carbon with x being an integer from 1-10 and R, represents hydrogen with y being an integer equal to two times the value of x plus two, and a suitable buffer. [30] In accordance with an embodiment of the present invention, a method comprising enantioselective enzymatic reduction of an arylketone represented by the following Chemical Formula 2 and the carbamation of an alcohol compound represented by the following Chemical Formula 3 is provided for the preparation of carbamic acid (R)- 1 -aryl-2-tetrazolyl-ethyl ester, represented by the following Chemical Formula 1. [31] [Chemical Formula 1] [32] WO 2011/046380 PCT/KR2010/007069 0

H

2 N 0 N -A1 N RI A 2 / R1 2 R2 [33] [Chemical Formula 2] [34] 0 N -:A, R1 NA 2 R2 [35] [Chemical Formula 3] [36] OH N--A I \N

A

2 R 1 2 R2 [37] wherein, [38] R 1 and R 2 are independently selected from a group consisting of hydrogen, halogen, perfluoroalkyl, alkyl of 1 to 8 carbon atoms, thioalkoxy of 1 to 8 carbon atoms, and alkoxy of 1 to 8 carbon atoms; and [39] one of A 1 and A2 is CH with the other being N. [40] The arylketone of Chemical Formula 2, useful as a starting material in the preparation method of the present invention, may be synthesized by, for example, a substitution reaction between the arylketone of Chemical Formula 4 and the tetrazole of Chemical Formula 5: [41] [Chemical Formula 4] [42] WO 2011/046380 PCT/KR2010/007069 0 R1 R2 [43] [Chemical Formula 5] [44] H N N. N N-N [45] wherein, [46] R, and R 2 are as defined above; and [47] X is a leaving group such as a halide or sulfonate. [48] [49] An economical advantage is given to the synthesis of the arylketone of Chemical Formula 2 from the compounds represented by Chemical Formulas 4 and 5 because they are commercially available, cheap compounds. In addition, the substitution reaction can be carried out in relatively mild conditions, compared to the ring-opening reaction between (R)-2-aryl-oxirane and tetrazole. The method according to the present invention is therefore certain of process safety although employing potentially explosive tetrazole, and ensures high production yield and easy purification, with the production of no unnecessary positional isomers at benzyl positions. [50] The arylketone represented by Chemical Formula 2 which can be synthesized by the substitution reaction with tetrazole may be in a mixture of positional isomers including IN arylketone of the following Chemical Formula 2a and 2N arylketone of the following Chemical Formula 2b, which can be isolated and purified through com mercially available crystallization. [51] [Chemical Formula 2a] [52] WO 2011/046380 PCT/KR2010/007069 0N N \N R1 'N [53] [Chemical Formula 2b] [54] O N N R1 NN R2 [55] The crystallization useful in the present invention may comprise adding a solu bilizing agent to the product of the substitution reaction, that is, a mixture of the po sitional isomers, and then adding a precipitating agent. Optionally, the crystallization may further comprise, after the precipitation, filtrating the precipitate, concentrating the filtrate and adding an additional precipitating agent. [56] Illustrative, non-limiting examples of the solubilizing agent include acetone, ace tonitrile, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, and lower alcohols of 1 to 4 carbon atoms, or combinations thereof. The solubilizing agent may be used in an amount of from 0 to 20 ml (v/w) based on the weight (g) of the mixture of the positional isomers. As used herein, the addition of the solubilizing agent in an amount of zero ml (v/w) is intended to mean immediately adding the subsequent precipitating agent without dilution of the filtrate. [57] Examples of the precipitating agent include water, C1 -C4 lower alcohol, diethylether, pentane, hexane, cyclohexane, heptane or combinations thereof, but are not limited thereto. The precipitating agent may be slowly added in an amount of from zero to 40 ml (v/w) based on the weight (g) of the mixture of positional isomers. As used herein, theaddition of the precipitating agent in an amount of zero ml is intended to mean leaving or cooling to yield precipitates without the addition of the precipitating agent. [58] The filtration of the precipitates thus obtained by the addition of the precipitating agent yields the iN arylketone of Chemical Formula 2a as a crystal with high purity. [59] On the other hand, the filtrate thus obtained after the filtration step may be con centrated to increase the ratio of the precipitating agent to the solubilizing agent, thereby yielding the 2N arylketone of Chemical Formula 2b with high purity. Con- WO 2011/046380 PCT/KR2010/007069 centration ratio of the filtrate can be suitably determined by those of ordinary skill in the art. For example, concentration is carried until the solvent is totally removed off, then the solubilizing agent and the precipitating agent are added as mentioned above. [60] Unlike column chromatography, this crystallization may be commercially used without much difficulty. [61] [62] The enantioselective enzymatic reduction allows for the conversion of the arylketone of Chemical Formula 2 into the alcohol compound with (R)-configuration, represented by the following Chemical Formula 3. [63] [Chemical Formula 2] [64] 0 N _A, N R1 N 2 R2 [65] [Chemical Formula 3] [66] OH N-A I \N N, //

A

2 R2 fy [67] wherein, [68] R 1 and R 2 are independently selected from a group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and alkoxy of 1 to 8 carbon atoms; and [69] one of A 1 and A2 is CH with the other being N. [70] The enantioselective enzymatic reduction may be performed using an oxidoreductase enzyme in suspension in the reaction mixture, or immobilized in a conventional manner. The enzyme may be utilized in a completely purified state, in a partially purified state, or in microbial cells where it was expressed. The cells themselves may be in a native state, a permeabilized state or a lysed state. It will be appreciated by those of ordinary skill in the art that use of the enzyme in the cells is preferred for the WO 2011/046380 PCT/KR2010/007069 practice of the process of the invention since it represents a significant savings in cost. Most preferably, the enzyme is expressed in E. coli and used as a suspension of native cells. [71] The process of enzymatic reduction of the aryl ketone compounds of Formula 2 can be performed in a reaction mixture comprising said compound of Formula 2, an oxi doreductase, NADH or NADPH as a cofactor, a cosubstrate and a suitable buffer wherein the oxidoreductase comprises an amino acid sequence having at least 60% of the amino acids are identical with one of the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4. [72] It has been found that polypeptides comprising one of amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4 or a polypeptides comprising an amino sequence which is identical by at least 60 %, preferably at least 90% to one of the amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4 and possessing oxidoreductase activity can be used for reducing the compound of Formula 2 to the compound of Formula 3 (R-configuration)with high conversion and high enatiomeric selectivity. The enantiomeric excess of the R-alcohol formed in the enantioselective enzymatic is at least about 89%, preferably at least about 95% and most preferably at least about 99%. [73] The organism producing the oxidoreductase polypeptides useful in the enantios elective enzymatic reduction may be a wild strain or a variant and is preferably selected from Candida magnolia, Candida vaccinii, and Oryctolagus cuniculus. Yeast of the Candida genus is preferred for producing the oxidoreductase enzymes utilized in the present process. Derivatives of the polypeptides are those having at least sixty percent homology with the SEQ IDs given above and possessing oxidoreductase activity. Those skilled in the art are aware that there are systems and technology available to accurately determine sequence homology. [74] A polypeptide comprising SEQ ID NO: 1 may be encoded by a DNA sequence SEQ ID NO:5 which is obtainable, for example, from the organism Oryctolagus cuniculus deposited under the conditions of the Budapest Treaty with the Deutsche Sammlung fur Mikroorganismen und Zellkulturen, Mascheroder Weg 1b, 38124 under the number DSMZ 22167, specifically from rabbit DSMZ 22167, or by a nucleic acid sequence that hybridizes therewith. A polypeptide comprising SEQ ID NO:2 may be encoded by a DNA sequence SEQ ID NO:6 which is obtainable, for example, from the organism Candida magnoliae DSMZ 22052, or by a nucleic acid sequence that hybridizes therewith. [75] A polypeptide comprising SEQ ID NO:3 may be encoded by a DNA sequence SEQ ID NO:7, which is obtainable, for example, from the organism Candida vaccinii CBS7318, or by a nucleic acid sequence that hybridizes therewith. A polypeptide WO 2011/046380 PCT/KR2010/007069 comprising SEQ ID NO:4 may be encoded by a DNA sequence SEQ ID NO:8, which is obtainable, for example, from the organism Candida nagnoliae CBS6396, or by a nucleic acid sequence that hybridizes therewith. [76] The oxidoreductase having one of polypeptide sequences mentioned above is obtained in useable quantities by conventional procedures recognized by those skilled in the art. A polynucleotide coding for the amino acid sequence is cloned into a suitable vector and thereafter introduced into a host organism capable of expressing the gene coding for the sequence. Microorganisms susceptible of transforming to become capable of expressing of such a peptide are well known in the art. A preferred mi croorganism is Escherichia coli. As stated above, the oxidoreductase expressed by transformed E. coli may be extracted from the E. coli cells and partially or completely purified for use in the process, or may be utilized in the cells themselves which may be in a native, permeabilized or lysed state. A preferred embodiment of the enantios elective enzymatic reduction utilizes a suspension of the oxidoreductase as cells in the native state. Any of these forms may be utilized in the free or immobilized form. [77] The reduction reaction may be carried out in a single phase system having the cells containing the enzyme suspended therein. Alternatively, the reaction may be performed in a two-phase aqueous/organic solvent system as described in U. S. Patent Application Publication No. 2009/0017510 and U. S. Patent No. 7,371,903. The reaction may be carried out as a conventional batch reaction, or as a continuous process. It will be appreciated that one of the significant advantages of the enantios elective enzymatic reduction for commercial applications is that it is amenable to continuous operation. [78] The reaction mixture preferably contains from about 35 g to 350 g of cells per kg of reactant added. The suspension is the aqueous portion of the reaction mixture which also contains a buffer, for example a TEA (triethanolamine), phosphate, Tris/HCl or glycine buffer. The buffer may additionally comprise ions for the stabilization of the enzyme, for example, a source of magnesium ions. Additional additives that may be present in the buffer for stabilizing the enzymes may include a polyol, such as glycerol, sorbitols and the like, sulfur compounds, such as 1,4-DL-dithiothreitol, glutathione, cysteine or the like, amino acids and peptides, or detergents, such as DMSO. A preferred stabilizer for the enzyme is a polyol, particularly glycerol, which may be present in from about 10% to 80% by weight, preferably about 50% by weight, based on the weight of the cell suspension. [79] The enantioselective enzymatic reduction process is advantageously carried out using a coupled substrate principle wherein the reaction mixture utilizes a cosubstrate for the regeneration of the cofactor, or coenzyme, which functions to provide hydrogen for the reduction of the arylketone substrate. The cofactor is preferably nicotineamide adenine WO 2011/046380 PCT/KR2010/007069 dinucleotide phosphate (NADP) or nicotineamide adenine dinucleotide (NAD), which are utilized in the reduced state, i.e. NADPH or NADH, respectively. The cofactor is present in the reaction mixture in a concentration of from about 0.01 mM to 5 mM, preferably 0.05 mM to 0.5 mM. In the reaction, the cosubstrate functions by being oxidized in the regeneration of the NADPH or NADH cofactor. The cosubstrate is a secondary alcohol represented by the Formula R.RCHOH, wherein R. represents carbon with x being an integer from 1-10 and R, represents hydrogen with y being an integer equal to two times the value of x plus two. Examples of suitable cosubstrates include 2-propanol, 2-butanol 4-methyl-2-pentanol, 2-pentanol, 2-heptanol, 2-octanol and the like. A preferred cosubstrate is 2-butanol. The cosubstrate is present in the reaction mixture in from about 10% to 80 % by volume, preferably from about 40% to 60 % by volume, most preferably about 50% by volume. [80] The oxidized cofactor formed during the reduction of the arylketone is regenerated by oxidation of the cosubstate, which also can be catalyzed by the oxidoreductase. Thus, a particular economic advantage of the present process is that the oxidoreductase affects both reduction of the arylketone of Formula 1 and oxidation of the cosubstrate, therefore no further enzyme has to be used for cofactor regeneration. It is also within the scope of the present invention to add another enzyme to the reaction mixture for cofactor regeneration in order to enhance the rate of reduction of the aryl ketone. [81] In a further embodiment, an organic solvent that is not involved in the regeneration of the cofactor may be added to the reaction mixture and the reduction process performed in aqueous organic 2-phase system. Examples of such solvents include, without intended limitation, diethyl ether, tertiary butyl methyl ether, diisopropyl ether, dibutyl ether, ethyl acetate, butyl acetate, heptane, hexane or cyclohexane. Such a solvent may be present in from about 1% to 50% by volume based on the volume of the reaction mixture. [82] The amount of the arylketone substrate in the reaction mixture is preferably greater than about 0.1% by weight and may be increased to about 50% by weight, with a preferred concentration being from about 5 to 30% by weight. The amount of the substrate will vary depending on the purity thereof since the process may be carried out with the substrate in a purified state or as raw product containing varying amounts and types of impurities. The pH of the reaction mixture after the addition of all components will be in the range of 5 to 10, preferably from 7 to 9, and optimally about pH 8. The enzymatic reduction according to the present invention is carried out at a temperature of from about 10-45 C., preferably from about 20-40'C., most preferably from about 25-35"C. [83] The enantioselective reduction process is cost-effective and environment-friendly in addition to providing the alcohols of Formula 3 in high yield and very high enantiose- WO 2011/046380 PCT/KR2010/007069 lectivity. Thus, an alcohol compound with an (R)-configuration of high optical purity can be obtained in the presence of the enzyme under the above-mentioned reaction conditions within from about 12 to 96 hours, preferably from about 24 to 48 hours. During the incubation, the pH of the mixture is maintained within the ranges given above by periodic testing and the addition of a conventional acidic or basic reagent, for example sodium carbonate and sodium hydroxide, respectively. The efficiency of the enantioselective enzymatic reduction can be expressed by the total turnover number (TTN) which is the moles of the chiral alcohol of Formula 2 produced per mole of cofactor initially added. The TTN of the enantioselective enzymatic reduction is from about 102 to 105, preferably >103. [84] When the alcohol compound obtained through the enantioselective enzymatic reduction exists as a positional isomer mixture of 1N alcohol of Chemical Formula 3a and 2N alcohol of Chemical Formula 3b, it can be isolated and purified into individual positional isomers of high purity by crystallization: [85] [Chemical Formula 3a] [86] O H N, N N R1-N R2 [87] [Chemical Formula 3b] [88] OH N-: N R1 N R2 [89] The crystallization may comprise adding a solubilizing agent to the positional isomer mixture resulting from the reduction; and adding a precipitating agent, and optionally filtering the precipitate; and concentrating the filtrate and adding an additional pre cipitating agent. [90] Examples of the solubilizing agent useful in the crystallization include acetone, ace tonitrile, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, WO 2011/046380 PCT/KR2010/007069 lower alcohol of 1 to 4 carbon atoms, and a mixture thereof, but are not limited thereto. The solubilizing agent may be added in an amount of zero to 20 ml (v/w) based on the weight (g) of the positional isomer mixture. [91] Non-limiting examples of the precipitating agent include water, a lower alcohol of I to 4 carbon atoms, diethylether, pentane, hexane, cyclohexane, heptane, and a mixture thereof. The precipitating agent may be slowly added in an amount of from zero to 40 ml (v/w) based on the weight (g) of the positional isomer mixture. [92] Following the addition of the precipitating agent, filtration may yield 1N alcohol (3a) as a precipitate of high purity. [93] Furthermore, 2N alcohol (3b) can be obtained as a crystal form of very high purity by concentrating the filtrate and increasing the ratio of the precipitating agent to the solu bilizing agent. [94] These crystallization steps may be omitted when the positional isomers of arylketone of Chemical Formula 2 are already isolated and purified. [95] The introduction of a carbomoyl moiety into the alcohol compound with (R)-configuration of Chemical Formula 3 leads to carbamate with (R)-configuration, represented by Chemical Formula 1: [96] [Chemical Formula 3] [97] OH N A \N N //

A

2 R1 2 R2 [98] [Chemical Formula 1] [99] 0 H N 0 N~ 1 2 A N R1 2 R2 [100] wherein, [101] R 1 and R 2 are independently selected from a group consisting of hydrogen, halogen, WO 2011/046380 PCT/KR2010/007069 perfluoroalkyl, an alkyl of I to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; and one of A 1 and A 2 is CH with the other being N. [102] In the carbamation step, for example, inorganic cyanate-organic acid, isocyanate water, or carbonyl compound-ammonia may be employed to introduce a carbamoyl moiety. [103] For the carbamation with inorganic cyanate-organic acid, the alcohol compound with (R)-configuration of Chemical Formula 3 is dissolved in an organic solvent, for example, diethylether, tetrahydrofuran, 1,4-dioxane, acetonitrile, dichloromethane, chloroform or a mixture thereof, and mixed with 1 to 4 equivalents of inorganic cyanate such as sodium cyanate and organic acid, such as methane sulfonic acid or acetic acid, followed by reacting at about -10 to 70'C. [104] With regard to use of the isocyanate-water, 1 to 4 equivalents of isocyanate, for example, chlorosulfonic isocyanate, trichloroacetyl isocyanate, trimethylsilyl isocyanate, are added to a solution of the alcohol compound with (R)-configuration of Chemical Formula 3 in an organic solvent, for example, diethylether, tetrahydrofuran, 1,4-dioxane, acetonitrile, dichloromethane, chloroform or a mixture thereof, and reacted at about -50 to 40'C. Subsequently, without purification, 1 to 20 equivalents of water are added to induce hydrolysis. [105] With regard to use of the carbonyl compound-ammonia, 1 to 4 equivalents of a carbonyl compound, for example, 1,1 'carbonyldiimidazole, carbamoly chloride, disuccinyl carbonate, phosgene, triphosgene, or chloroformate, are added to a solution of the alcohol compound with (R)-configuration of Chemical Formula 3 in an organic solvent, for example, diethylether, tetrahydrofuran, 1,4-dioxane, acetonitrile, dichloromethane, chloroform or a mixture thereof, and reacted at about -10 to 70'C, followed by adding 1 to 10 equivalents of ammonia without purification. [106] After the carbamation, the carbamate compound of Chemical Formula 1 thus obtained may be purified to higher optical and chemical purity through the following crystallization. The crystallization comprises adding a solubilizing agent to the product of the carbamation; and then adding a precipitating agent, and optionally filtering the precipitate and adding an additional precipitating agent. For pharmaceutical use, it is preferable that there is always a final purification of the carbamated product before use, but that there can be a crystallization step earlier in the process. [107] Non-limiting examples of the solubilizing agent include acetone, acetonitrile, tetrahy drofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, lower alcohol of 1 to 4 carbon atoms, and a mixture thereof. Based on the weight (g) of the reaction product, the solubilizing agent may be used in an amount of from zero to 20 ml (v/w). [108] Non-limiting examples of the precipitating agent include water, lower alcohols of I WO 2011/046380 PCT/KR2010/007069 to 4 carbon atoms, diethylether, pentane, hexane, cyclohexane, heptane and a mixture thereof. Based on the weight (g) of the reaction product, the precipitating agent may be slowly added in an amount of from zero to 40 ml (v/w). [109] Comprising enantioselective enzymatic reduction, the method of the present invention can provide optically high pure carbamate compounds. In addition, the mild reaction conditions which the method of the present invention requires ensure process safety. Furthermore, the crystallization step applicable to large-scale production before or after the enantioselective enzymatic reduction or after the carbamation results in a higher chemical purity of the carbamate compounds. The carbamate compounds prepared according to the present invention are very useful in the treatment of CNS disorders such as convulsion. Mode for the Invention [110] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as limiting the present invention. [111] [112] Preparation Example 1: Preparation of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol- 1-yl)ethan-1-one [113] [114] To a suspension of 2-bromo-2'-chloroacetophenone (228.3 g, 0.978 mol) and potassium carbonate (161.6 g, 1.170 mol) in acetonitrile (2000 mL) was added a 35 w/ w% IH-tetrazole dimethylformamide solution (215.1 g, 1.080 mol) at room tem perature. These reactants were stirred for 2 h at 45'C and distilled under reduced pressure to remove about 1500 mL of the solvent. The concentrate was diluted in ethyl acetate (2000 mL) and washed with 10% brine (3 x 2000 mL). The organic layer thus separated was distilled under reduced pressure to afford 216.4 g of an oily solid residue. To a solution of the solid residue in ethyl acetate (432 mL) was slowly added heptane (600 mL). The precipitate thus formed was filtered at room temperature and washed to yield 90.1 g (0.405 mol) of 1- (2-chlorophenyl)-2- (1,2,3,4-tetrazol- 1 -yl)ethan- 1-one (hereinafter referred to as 1 N ketone ). [115] 'H-NMR(CDCl 3 ) 8.87(s, lH), d7.77(d, lH), d7.39-7.62(m, 3H), d5.98(s, 2H) [116] [117] Preparation Example 2: Preparation of 1- (2-chlorophenyl)-2- (1,2,3,4-tetrazol-2-yl)ethan- 1-one [118] [119] After the filtration of Preparation Example 1, the filtrate was concentrated and WO 2011/046380 PCT/KR2010/007069 dissolved in isopropanol (100 mL), and to which heptane (400 mL) was then added to complete the crystallization. Filtering and washing at 5'C afforded 1- (2-chlorophenyl)-2- (1,2,3,4-tetrazol-2-yl)ethan- 1-one (hereinafter referred to as "2N ketone") as a solid. 94.7 g (0.425 mol). [120] 1 H-NMR(CDCl 3 ) d8.62(s, 1H), d7.72(d, 1H), d7.35-7.55(m, 3H), d6.17(s, 2H) [121] [122] PREPARATION EXAMPLE 3: Preparation of Alcohol Compound of (R)-Configuration by enantioselective enzymatic reduction via various oxidoreductases [123] The following four solutions were prepared as follows: [124] [125] Enzyme Solution 1 [126] Competent Escherichia coli StarBL21(De3) cells (Invitrogen) were transformed with the expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 1. The Escherichia coli colonies transformed with the resulting expression constructs were then cultivated in 200 mL of LB medium (1% tryptone, 0.5 % yeast and 1% sodium chloride) with 50 micrograms/mL of ampicillin or 40 micrograms/mL of kanamycin, respectively, until an optical density of 0.5, measured at 550 nm, was achieved. The expression of the desired recombinant protein was induced by the addition of isopropy lthiogalactoside (IPTG) to a concentration of 0.1 mM. After 16 hours of induction at 25 'C and 220 rpm, the cells were harvested and frozen at -20 'C. In the preparation of the enzyme solutions, 30 g of cells were resuspended in 150 mL of triethanolamine buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) and homogenized in a high pressure homogenizer. The resultant enzyme solution was mixed with 150 mL glycerol and stored at -20'C. [127] [128] Enzyme Solution 2 [129] RB791 cells (E.coli genetic stock, Yale, USA) were transformed with the expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 2. The Escherichia coli colonies transformed with the resulting expression constructs were then cultivated in 200 mL of LB medium (1% tryptone, 0.5 % yeast and 1% sodium chloride) with 50 micrograms/mL of ampicillin or 40 micrograms/mL of kanamycin, respectively, until an optical density of 0.5, measured at 550 nm, was achieved. The expression of the desired recombinant protein was induced by the addition of isopropylthiogalactoside (IPTG) to a concentration of 0.1 mM. After 16 hours of induction at 25'C and 220 rpm, the cells were harvested and frozen at -20'C. In the preparation of the enzyme solutions, 30 g of cells were resuspended in 150 mL of triethanolamine buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) and homogenized in a high pressure ho mogenizer. The resultant enzyme solution was mixed with 150 mL glycerol and stored WO 2011/046380 PCT/KR2010/007069 at -20"C. [130] [131] Enzyme Solution 3 [132] Enzyme solutions 3 was prepared in the same manner as described in Enzyme solution 1 except that expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 3 instead of expression constructs pET2 1-MIX coding for oxidoreductase SEQ ID NO 1 was used. [133] [134] Enzyme Solution 4 [135] Enzyme solutions 4 was prepared in the same manner as described for enzyme solution 2 except that expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 4 instead of expression constructs pET21-MIX coding for oxidoreductase SEQ ID NO 2 was used. [136] [137] Different oxidoreductases contained in each enzyme solutions 1 to 4 were examined as follows for the conversion of 1- (2-chlorophenyl)-2- (1,2,3,4-tetrazol- 1 -yl)ethan- 1-one (1N ketone) and 1- (2-chlorophenyl)-2- (1,2,3,4-tetrazol-2-yl)ethan- 1-one (2N ketone) to the corre sponding 1- (2-chlorophenyl)-2-(1,2,3,4-tetrazol- 1 -yl)ethan- 1 -ol (hereinafter, referred to as IN alcohol ) and 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (hereinafter, referred to as "2N alcohol"), respectively. [138] [139] Reaction batch A [140] 160 pi buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) [141] 100 af NADPH (40 mg/ml) [142] 40 af 2-propanol [143] 50 u enzyme solution 1 [144] 2 mg 1N ketone or 2N ketone [145] [146] Reaction batch B [147] 160 a0 buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) [148] 100 p NADPH (40 mg/ml) [149] 40 aft 2-propanol [150] 50 f enzyme solution 2 [151] 2 mg 1N ketone or 2N ketone [152] [153] Reaction batch C [154] 350 ,a buffer (TEA 100 nM, 2 mM MgCl2, 10% glycerol, pH 8) WO 2011/046380 PCT/KR2010/007069 [155] 0,05 mg NADP [156] 50 ue enzyme solution 3 [157] 10 mg 1N ketone or 2N ketone [158] 250 pft 4-methyl-2-pentanol [159] 50 2, enzyme (oxidoreductase from Thermoanerobiun brockii) solution for re generation of cofactor [160] [161] Reaction batch D [162] 350 , buffer (TEA 100 nM, 2 mM MgCI2, 10% glycerol, pH 8) [163] 0,05 mg NADP [164] 50 aft enzyme solution 4 [165] 10 mg iN ketone or 2N ketone [166] 250 ,0 4-methyl-2-pentanol [167] 50 af enzyme (oxidoreductase from The rmoanerobiun brockii) solution for re generation of cofactor [168] After 24h of incubating each reaction batch A, B, C and D, I mL of acetonitrile was added to each reaction batch which was centrifuged and transferred into a HPLC analysis vessel for enantiomeric excess and conversion. Conversion and ee-value of products are listed in Table 1 below calculated using the following equations: [169] Conversion Rate (%) = [(Area of Product)/(Area of Reactant + Area of Product)]x100 [170] ee-value(%) = [(Area of R-Configuration - Area of S-Configuration)/(Area of R Configuration + Area of S-Configuration)] x 100 [171] Table 1 [Table 1] ee-values Conversion %ee(enantiomer) (%of reduced ketone) R-2N Alcohol,2b R-lN Alcohol, 2a Reaction batch A >98 89(R) >99(R) Reaction batch B >98 >99(R) >99(R) Reaction batch C >98 95(R) >99(R) Reaction batch D >98 98(R) 95(R) [172] PREPARATION EXAMPLE 4: Enzymatic reduction via oxidoreductase SEQ NO: 2 [173] For the conversion of 1N/2N ketone to R- 1N/R-2N alcohol, 30yA of the enzyme solution 2 containing the oxidoreductase SEQ NO: 2 were added to a mixture of 300y2 of a buffer (100 mM TEA, pH 8, 1mM MgCl2, 10% glycerol), 100mg of a mixture of WO 2011/046380 PCT/KR2010/007069 IN ketone and 2N ketone (1N:2N=14%:86%), 0.04mg NADP and 300y2 2-butanol. The reaction mixture was incubated at room temperature under constant thorough mixing. After 48 hours, more than 98% of the ketones were reduced to an alcohol mixture of the following composition(R-2N alcohol 80%; S-2N alcohol 0%; R-IN alcohol 20%, S-1N alcohol 0%; IN ketone 0%; 2N ketone 0%). [174] After general work up and recrystallization with ethyl acetate/hexane, optically pure alcohols were obtained as below: [175] (R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-1-yl)ethan-1-ol (1N alcohol) [176] 1 H-NMR(CDCl 3 ) d8.74(s, IH), d7.21-7.63(m, 4H), d5.57(m, IH), d4.90(d, IH), d4.50(d, 1H), d3.18(d, 1H); [177] (R)-1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (2N alcohol) [178] 1 H-NMR(CDCl 3 ) d8.55(s, 1H), d7.28-7.66(m, 4H), d5.73(d, 1H), d4.98(d, 1H), d4.83(d, 1H), d3.38(br, 1H). [179] [180] Preparation of Carbamate [181] [182] Preparation Example 5: Preparation of Carbamic Acid (R)- 1-(2-Chlorophenyl)-2-(tetrazol-2-yl)ethyl ester [183] 50ml of the enzyme solution 2 containing the oxidoreductase SEQ NO: 2 were added to a mixture of 250ml of a buffer (100 mM TEA, pH 8, 1mM MgCl2, 10% glycerol), 50g (225mmol) of 1-(2-chlorophenyl)-2-( 1,2,3,4-tetrazol-2-yl)ethan-1-one(2N ketone), 4mg NAD, 300 ml of 2-propanol and 150mL of butyl acetate. The reaction mixture was stirred at room temperature. After 48 hours more than 98% of 2N ketone was reduced to corresponding (R)- 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (R-2N alcohol) with >99%ee values. To this resulting mixture, 500mL of ethyl acetate was added. After being separated, the organic layer thus formed was washed with 10% brine (3 x 500mL). The organic layer thus formed was dried over magnesium sulfate and filtered and the filtrate was distilled under reduced pressure to give 50.4g (224 mmol) of 1-(2-chlorophenyl)-2-(1,2,3,4-tetrazol-2-yl)ethan-1-ol (R-2N alcohol, optical purity 99.9%) as an oily residue. To this resulting crude product, 450mL of tetrahy drofuran was added. After cooling to -15'C, 38g (267mmol) of chlorosulfonyl isocyanate was slowly added and stirred at -10 C for 2 h. The slow addition of water induced termination of the reaction. The resulting solution was concentrated under reduced pressure until about 300 mL of the solvent was removed. The concentrate was diluted with 600mL of ethyl acetate and washed with 10% brine (3 x 500 mL). The organic layer was concentrated under reduced pressure and the concentrate was dissolved in isopropanol (90 mL) to which heptane (180 mL) was slowly added, leading to the completion of crystallization. The precipitate thus obtained was filtered WO 2011/046380 PCT/KR2010/007069 and washed to afford 51.8 g (194 mmol) of carbamic acid (R)- 1-(2-chlorophenyl)-2-(tetrazol-2-yl)ethyl ester (optical purity 99.9%). [184] 'H-NMR(Acetone-d 6 ) d8.74(s, 1H), d7.38-7.54(m, 4H), d6.59(m, 1H), d6.16(Br, 2H), d4.90(d, IH), d5.09(m, 2H) [185] [186] As described hitherto, carbamate compounds with high optical and chemical purity can be produced with an economical benefit in accordance with the present invention. [187] [188] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims. Sequence Listing Free Text [189] AMINO ACID SEQUENCES [190] [191] SEQ ID NO 1: Oryctolagus cuniculus from rabbit DSMZ 22167 [192] 1 massgvtrrd plankvaivt astdgiglai arrlaqdgah vvissrkqqn vdravaalqa 61 eglsvtgtvc hvgkaedrer lvatalnlhg gidilvsnaa vnpffgklmd vteevwdkil 121 dinvkamalm tkavvpemek rgggsvviva siaafhpfsg lgpynvskta lvgltknIlal 181 elaaqnirvn clapglikts fskalwedka qeeniiqklr irrlgkpeec agivsflcse 241 dasyitgetv vvaggapsrl [193] SEQ ID NO 2: Candida magnoliae DSMZ 22052 protein sequence carbonyl reductase [194] 1 msatsnalit qasrgmgeat aiklalegys vtlasrgieg Inaikeklpi vkkgqqhyvw 61 qldlsdieaa stfkgapIpa ssydvffsna gvvdfapfad qsetaqkdlf tvnllspvaI 121 tktivkaiad kpretpahii ftssivgirg vpnvavysat kgaidsfars larefgpkni 181 hvncvnpgtt rtentkgvdl aatgdvpikg wievdaiada vlflikskni tgqslvvdng 241 fgv [195] SEQ ID NO 3: Candida vaccinii CBS7318 protein sequence carbonyl reductase [196] 1 mrstpnalvt ggsrgigaaa aiklaeagys vtlasrgidk lnevkakipv vkqgqehhvw 61 qldlsdvqaa lefkqaplpa skydlfvsna gvatfsptae hddkdwqnii avnltspiai 121 tkalvkavge rsndnpfqia fissaaalrg vpqtavysat kagldgftrs lakelgpkgi 181 hvnivhpgwt qtemtagvde prdtpipgwi qpeaiaeaiv ylaksknitg tnivvdnglt 241 i [197] SEQ ID NO 4: Candida ma gnoliae CBS6396 protein sequence carbonyl reductase WO 2011/046380 PCT/KR2010/007069 [198] 1 mnalvtggsr gigeaiatkl aedgysvtia srgidqlnkv kakipvvreg qthhvwqldl 61 sdaeaassfk gaplpassyd vlvnnagvtd pspiakqsds eihklfsvnl lspvaltkty 121 vqavtgkpre tpahiifiss gvairgypnv avysatksgl dgfmrslare lgpegvhvnt 181 vspgltktem asgvslddfp pspiggwigp eaiadavryl vksknitgti lsvdngitv [199] NUCLEIC ACID SEQUENCES [200] SEQ ID NO 5: Oryctolagus cuniculus from rabbit DSMZ 22167 [201] 1 atggettcat ctggcgtaac acgccgtgat ccgctggcca acaaagtcge tattgtcact 61 gcgtcgaccg atggcatcgg actggcgatt gcgcgtcgcc ttgctcagga cggggctcac 121 gtggtaatct cttcgcgtaa acagcaaaat gtagatcgtg ccgttgctge cctgcaagca 181 gaaggtctgt ccgtaactgg tactgtgtgc catgtcggga aagccgagga ccgtgaacgt 241 ctggttgcga cggcccttaa tcttcatggc ggtatcgata tcctggtgag taacgcggcc 301 gtcaatecgt ttttcggtaa gttaatggac gtcaccgaag aggtgtggga taaaattctg 361 gacatcaacg tgaaagcaat ggcgttgatg accaaagcgg tggttccaga aatggaaaaa 421 cgeggtgggg getcagttgt cattgtggcc agcattgeag cctttaatcc atttagcgge 481 ttaggtccgt acaatgtgag taaaacggca ttggttggcc tgaccaagaa cctggcattg 541 gagttagcag cgcagaacat tcgtgttaac tgtttagcgc cgggcctgat taagacatca 601 ttcagtaagg cactgtggga ggataaagct caggaggaaa atatcattca gaaactgcgt 661 attcgccgtc tgggaaaacc ggaagaatgt gcaggtateg ttagetttet gtgctetgaa 721 gatgcgtcet atattacggg tgaaaccgta gtggttgccg gcggagegcc gagccgcctg [202] SEQ ID NO 6: Candida magnoliae DSMZ 22052 nucleic acid sequence carbonyl reductase [203] 1 atgtctgcta cttcgaacgc tcttatcact ggtgccagcc gcggaatggg cgaggccaca 61 gctattaagc ttgcccttga ggggtacagc gtcacccttg catcacgcgg tattgagcag 121 ctcaatgcca tcaaggaaaa actacccatc gtgaagaagg gccagcagca ctacgtttgg 181 cagctcgatc ttagtgacat cgaggcggct tccaccttca aqggggctcc tctgcctgcc 241 agcagctacg acgtgttctt cagcaacgcc ggtgtggtgg actttgctcc gttcgcagac 301 caaagcgaga ctgcgcaaaa ggacctgttc acggttaacc tgctgtcgcc tgttgcgttg 361 accaagacca ttgttaaggc catcgccgac aagccccgcg agacgcctgc tcacattatc 421 ttcacctcgt ccattgtcgg aattcgcggt gttcccaacg tggcggtcta cagcgccacc 481 aagggcgcga ttgacagctt tgccgcctcg cttgctcgtg agttcggtcc caagaacatc 541 cacgttaact gcgtgaaccc gggcacgacg cqcaccqaqa tgacaaaggg cgttgatctc 601 gcggctttcg gcgatgttcc tatcaaqggc tggatcgagg tcgatgcgat tgccgacgct 661 gtgctgtttt tgatcaagtc caagaacatc actqgccagt cgctcgttgt tgacaacgga 721 ttcqgtgttt aa [204] SEQ ID NO 7: Candida vaccinii CBS7318 nucleic acid sequence carbonyl reductase WO 2011/046380 PCT/KR2010/007069 [205] 1 atgaggtcga cacctaacgc ccttgtgact ggcggcagcc gcggcattgg cgcggccgct 61 gcaattaaac tcgccgaggc aggetacage gtgacgctcg cgtcgcgcgg tctcgacaag 121 ctcaacqagg tgaaggccaa gcttcctgtc gtgaagcagg gccaggagca ccatgtatgg 181 Cagcttgatc tcagcgacgt gcaggccgcg cLcgagttca agggcgcacc gctgcccgcg 241 agtaagtacg atttgtttgt ctcgaacgcc ggcgtggcta ctttctcgcc aacggctgag 301 catgacgaca aggactggca gaacattatt gccgtgaact tgacatcgcc cattgccatt 361 acgaaggcgc tcgttaaggc cgttggcgag cgctcaaacg ataacccgtt tcagatcgcg 421 ttcctgtcat cggcggccgc cctgcgcggt gtgccgcaga ccgctgttta cagcgctacg 481 aaggccggcc tcgacggctt cacgcgctcg ctcgccaagg agctcggccc aaagggcatc 541 catgtgaaca tcgtacaccc tggatggacg cagaccgaga tgactgcggg tgtagatgag 601 cctagggata cgcccatccc gggctggatc cagccggaag ccatcgccga ggccattgtg 661 tatctcgcga agtcaaagaa catcacggga acgaacatcg ttgtcgacaa cggcctgact 721 atttaa [206] [207] SEQ ID NO 8: Candida magnoliae CBS6396 nucleic acid sequence carbonyl reductase [208] 1 atgaacgctc tagtgaccgg tggtagccgt ggcattggcg aggcgatcec gaccaagctg 61 gccgaagatg gctacagcgt gacaatcgcc tcgcgcggaa tcgatcagct caacaaggta 121 aaggctaaac ttccggttgL gagggagggc cagacccacc acgtgLggca gcttgatttg 181 agcgacgccg aggccgcgtc gtccttcaag ggcgctcctt tgccagcaag cagctacgat 241 gtccttgtca acaacgccgg agtaacggat ccgagtccca ttgcgaagca gtcggatagc 301 gagattcaca agctgtttag cgtgaatctg ctgtcaccag ttgctttgac aaagacgtac 361 gtccaggcgg ttaccggaaa gcctcgtgag acgccagctc acattatttt tatctcgtca 421 ggcgttgcca ttcgaggcta cccaaacgtc gctqtatact cggctactaa gaCggctc 481 gacggtttca tgaggtctct ggcgcgcgag cttggccccq agggcgtcca tgtgaacact 541 gtcagcccgg gtctcaccaa aaccgagatg gccagcggcg tcagcctcga cgacttcccg 601 ccatcgccga ttgggggctg gatccagccc gaggccatcg ctgatgcagt gaggtacctg 661 gtgaagtcga agaacatcac aggcacgatt ctgtcagttg acaacggaat cacggtttaa

Claims

1. A method for preparing carbamic acid aryl-2-tetrazolyl ethyl ester, represented by Chemical Formula 1, comprising: subjecting an arylketone, represented by Chemical Formula 2, to enantioselective 5 enzymatic reduction to form an alcohol compound of (R)-configuration, represented by Chemical Formula 3; and carbamating said alcohol [Chemical Formula 1] 0 H 2 N 0 N Al N RO N- A, R- N R2 [Chemical Formula 2] ' N RIH N A R2N 10 R [Chemical Formula 3] OH N Al N N RI A R2 wherein R 1 and R 2 are independently selected from a group consisting of 15 hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; one of A 1 and A 2 is CH with the other being N; and 24 said enantioselective enzymatic reduction is carried out in a reaction mixture comprising said compound of Chemical Formula 2, an oxidoreductase having at least 60% homology with amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID 5 NO:4, NADH or NADPH as a cofactor that is oxidized during the reduction process and continuously regenerated, a cosubstrate comprising a secondary alcohol represented by the Formula RxRYCHOH, wherein Rx represents carbon with x being an integer from 1-10 and Ry represents hydrogen with y being an integer equal to 10 two times the value of x plus two, and a suitable buffer.

2. The method according to claim 1, wherein the oxidoreductase is encoded, respectively, by nucleic acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:8, or by a nucleic acid sequence that hybridizes to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or 15 SEQ ID NO:8.

3. The method according to claim 2, wherein the oxidoreductase can be isolated from Candida magnolia, Candida vaccinii or Oryctolagus cuniculus.

4. The method according to any one of claims 1 to 3, wherein the 20 oxidoreductase is present in reaction mixture in a completely purified state, a partially purified state, or in the microbial cells that expressed it.

5. The method according to claim 4, wherein the oxidoreductase is present in the microbial cells that expressed it, which cells are in a native, permeabilized or lysed state. 25

6. The method according to claim 5, wherein the microbial cells are transformed Escherichia coli cells.

7. The method according to any one of claims 1 to 6, wherein the regeneration of the oxidized cofactor results from the oxidation of said cosubstrate. 30

8. The method according to any one of claims 1 to 7, wherein said cosubstrate is a secondary alcohol selected from the group consisting of 2-propanol, 2-butanol, 2-pentanol, 4-methyl-2-pentanol, 2-heptanol and 2-octanol. 25

9. The method according to any one of claims 1 to 8, wherein said oxidoreductase affects both the reduction of the arylketone of Formula 2 and the oxidation of the cosubstrate.

10. The method according to any one of claims 1 to 9, wherein the 5 carbamating step is carried out by reacting the alcohol compound of (R)-configuration of Chemical Formula 3 with inorganic cyanate and an organic acid.

11. The method according to any one of claims 1 to 9, wherein the carbamating step is carried out by hydrolyzing a product resulting from 10 reaction between the alcohol compound of (R)-configuration of Chemical Formula 3 and an isocyanate compound selected from the group consisting of chlorosulfonic isocyanate, trichloroacetyl isocyanate and trimethylsilyl isocyanate.

12. The method according to any one of claims 1 to 9, wherein the 15 carbamating step is carried out by introducing ammonia into a product resulting from reaction between the alcohol compound of (R) configuration of Chemical Formula 3 and a carbonyl compound comprising 1,1' - carbodiimidazole, carbamoylhalide, disuccinyl carbonate, phosgene, triphosgene or chloroformate. 20

13. The method according to any one of claims 1 to 12, further comprising a crystallizing step after at least one of enantioselective enzymatic reduction step and the carbamating step.

14. The method according to claim 13, wherein the crystallizing step comprises: 25 adding to a reaction product a solubilizing agent selected from among acetone, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, a lower alcohol of 1 to 4 carbon atoms and a mixture thereof; and adding a precipitating agent thereto selected from the group consisting 30 of water, a lower alcohol of 1 to 4 carbon atoms, diethylether, pentane, hexane, cyclohexane, heptane and a mixture thereof. 26

15. The method according to any one of claims 1 to 14, additionally including the step of preparing the arylketone of Chemical Formula 2 is prepared by substitution reaction between an arylketone of the following Chemical Formula 4 with a tetrazole of the following 5 Chemical Formula 5: [Chemical Formula 4] 0 RI R2 [Chemical Formula 5] H N N N-N 10 wherein R 1 and R 2 are as defined in claim 1; and X is a leaving group selected from among a halide and a sulfonate.

16. The method according to claim 15, further comprising a crystallizing step comprising: 15 adding a solubilizing agent selected from among acetone, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane, chloroform, 1,4-dioxane, a lower alcohol of 1 to 4 carbon atoms and a mixture thereof to a product obtained by the substitution reaction; and adding a precipitating agent selected from water, a lower alcohol of 20 1 to 4 carbon atoms, diethylether, pentane, hexane, cyclohexane, heptanes and a mixture thereof.

17. A method for preparing an alcohol compound, represented by the following Chemical Formula 3, through the enantio selective enzymatic reduction of an arylketone, represented by the following Chemical 25 Formula 2: 27 [Chemical Formula 2] 0 N---Al A N R14N A2 [Chemical Formula 3] %H NNA 5 wherein, R 1 and R 2 are independently selected from a group consisting of hydrogen, halogen, perfluoroalkyl, an alkyl of 1 to 8 carbon atoms, a thioalkoxy of 1 to 8 carbon atoms, and an alkoxy of 1 to 8 carbon atoms; 10 one of A 1 and A 2 is CH with the other being N; and said reduction is carried out in a reaction mixture comprising said compound of Chemical Formula 2, an oxidoreductase having at least 60% homology with amino acid sequences SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO:4, NADH or NADPH as a 15 cofactor that is oxidized during the reduction process and continuously regenerated, a cosubstrate comprising a secondary alcohol represented by the Formula RxRYCHOH, wherein Rx represents carbon with x being an integer from 1-10 and Ry represents hydrogen with y being an integer equal to two times the value of x plus two, and a suitable buffer. 20

18. The method according to claim 17, wherein the oxidoreductase is encoded, respectively, by nucleic acid sequence SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:8, or by a nucleic acid sequence that hybridizes to SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO:8, respectively. 28

19. The method according to claim 17, wherein the oxidoreductase can be isolated from Candida magnolia, Candida vaccinii or Oryctolagus cuniculus.

20. A method for preparing carbamic acid aryl-2-tetrazolyl ether ester of the 5 Chemical Formula 1 as defined in claim 1, or a method of preparing an alcohol compound of Chemical Formula 3 as defined in claim 17, which method is substantially as herein described with reference to the Examples. SK Biopharmaceuticals Co., Ltd. 10 Patent Attorneys for the Applicant/Nominated Person SPRUSON & FERGUSON